Kazuyoshi Hashiguchi

Aberrant Gene Methylation in the Lymph Nodes Provides a Possible Marker for Diagnosing Micrometastasis in Gastric Cancer

BackgroundThis study was designed to determine whether gene methylation is a novel diagnostic marker for micrometastasis to the lymph nodes (LNs) in gastric cancer.MethodsThe gene methylation of CHFR, p16, RUNX3, E-cadherin, MGMT, hMLH1, and ABCG2 genes were analyzed in 49 primary gastric cancer tissues, corresponding to noncancerous tissues and matched LNs by quantitative methylation-specific PCR (q-MSP).ResultsCHFR, RUNX3, MGMT, and hMLH1 were more frequently methylated in primary cancer compared with the noncancerous mucosa. Further analyses investigated whether the methylation of the four cancer-specific genes was preserved in LN tissues using the 29 control cases, in which LN metastasis had been histologically confirmed. The methylation of both lesions (M/M pattern) in at least one gene, which was judged to be positive for cancer cells in LNs, was observed in 25 of 29 cases (86%). Quantitative RT-PCR (qRT-PCR) of CEA, CK19, and CK20 mRNA was conducted using the same samples. The mRNA expression of at least one of the three genes was observed in 100% of the specimens. The results of the control analysis were used to attempt to predict micrometastasis by q-MSP and qRT-PCR in the 20 test cases without histological LN metastasis. Six cases (30%) showed the M/M pattern in at least one of the four genes. Three of 20 cases (15%) exhibited both the M/M pattern and positive mRNA.ConclusionsThe methylation analysis revealed the clinical feasibility of detecting occult neoplastic cells in the regional LNs.

Expression of hypoxic marker CA IX is regulated by site-specific DNA methylation and is associated with the histology of gastric cancer.

The hypoxic marker carbonic anhydrase (CA) IX has been recognized as a tumor-associated protein and is essential for cancer development. However, because CA IX expression does not always correlate with hypoxia, its regulatory mechanism remains unclear. The objective of the present study was to clarify the role and regulation of CA IX expression in gastric cancer. The immunohistochemical expression of CA IX and hypoxia-inducible factor-1α was assessed in 77 patients with gastric cancer. A methylation-sensitive restriction enzyme method was used to quantify site-specific methylation at -74 bp in the CA9 promoter in tissue from patients with gastric cancer and in corresponding normal tissue. CA9 expression in cell lines was strongly dependent on methylation status but not hypoxic stimuli. In tissue from patients with gastric cancer, the quantity of methylation was significantly correlated with the protein expression (P = 0.003). Moreover, the methylation value was significantly lower in intestinal-type compared with diffuse-type cancer (P = 0.003). Compared with normal mucosa, intestinal-type cancer demonstrated significant hypomethylation, whereas diffuse-type cancer exhibited hypermethylation. In conclusion, expression of CA IX in gastric cancer is predominantly regulated by methylation of a single CpG rather than by hypoxia. Furthermore, epigenetic alterations in CA9 differ between the intestinal and diffuse types of gastric cancer.

HIF‐1α is an unfavorable determinant of relapse in gastric cancer patients who underwent curative surgery followed by adjuvant 5‐FU chemotherapy

Among several chemotherapeutic agents, 5‐fluorouracil (5‐FU) has been widely used as a key drug in adjuvant chemotherapy for gastric cancer. However, no reliable marker, which predicts the response to 5‐FU in an adjuvant setting, has been identified. Hypoxia‐induced drug resistance, via upregulation of HIF‐1α, is a major obstacle in the development of effective cancer therapy. However, few clinical studies have so far assessed the relationship between the HIF‐1α expression and the chemo‐resistance of gastric cancer patients in an adjuvant setting. We established 2 HIF‐1α knockdown gastric cancer cell lines in order to clarify the role of HIF‐1α in chemo‐resistance against 5‐FU. Furthermore, expression of HIF‐1α was immunohistochemically assessed in 91 resected specimens. Sixty‐four of 91 patients received 5‐FU adjuvant chemotherapy after surgery. HIF‐1α expression was associated with the significantly shorter relapse‐free survival and disease‐specific survival in the 64 patients of adjuvant group (p = 0.026, 0.014, respectively), but not in the 27 of surgery group. Multivariate analysis showed that HIF‐1α was an independent risk factor for relapse in 64 patients in the adjuvant group (p = 0.029). In conclusion, the current study confirmed, for the first time that HIF‐1α expression is an independent risk factor for relapse in high‐risk gastric cancer patients who underwent curative surgery followed by adjuvant 5‐FU chemotherapy. A favorable effect of 5‐FU might therefore be expected in patients that do not express HIF‐1α, whereas, other types of chemotherapy or additional treatments, such as HIF‐1α inhibitors, should be considered in patients that do express HIF‐1α.

Knockdown of hypoxia-inducible factor-1α accelerates peritoneal dissemination via the upregulation of MMP-1 expression in gastric cancer cell lines

This study was performed to clarify the role of hypoxia-inducible factor-1 α (HIF-1α) in the development of peritoneal dissemination in a xenograft mouse model of gastric cancer. HIF-1α knockdown (KD) and control (SC) gastric cancer cells, which were established using the MKN45 and MKN74 cell lines, were studied. The two paired cell lines were directly inoculated into the peritoneal cavity of nude mice. The number and the weight of disseminated nodules were compared between tumors generated from the KD and SC cells. In addition, the molecular mechanism was addressed through analysis of the expression levels of metastasis-related genes. The MKN45-KD cell line demonstrated significantly greater numbers of disseminated nodules and formed a larger tumor mass than the MKN45-SC cell line (p<0.05). MKN74-KD cells also tended to induce a greater number of nodules and to produce those with a heavier weight than the SC cells. An in vitro adhesion assay revealed differing results regarding the adhesion activity to extracellular matrix and monolayer mesothelium cells of the gastric cancer cells derived from the various parental cells. However, the expression of MMP-1 mRNA in the disseminated nodules was significantly increased in the KD cells compared to the SC cells derived from the two parental cell lines (p<0.01). An immunohisto-chemical study further demonstrated that there was stronger staining for MMP-1 in the MKN74-KD in comparison to MKN74-SC cells. Loss of HIF-1α may contribute to the development of aggressive peritoneal dissemination via the upregulation of MMP-1 in gastric cancer cells.

Abstract LB-376: Hypoxia-inducible factor-1 alpha is an unfavorable determinant of relapse in gastric cancer patients who underwent curative surgery followed by adjuvant 5-fluorouracil chemotherapy

Background: Among several chemotherapeutic agents, 5-Fluorouracil (5-FU) has been widely used as a key drug in adjuvant chemotherapy for gastric cancer. However, no reliable marker, which predicts the response to 5-FU in an adjuvant setting, has been identified. Hypoxia-induced drug resistance, via up-regulation of hypoxia-inducible factor (HIF) -1α, is a major obstacle in the development of effective cancer therapy. Despite the numerous investigations, few clinical studies have so far assessed the relationship between the HIF-1α expression and the chemo-resistance of gastric cancer patients in an adjuvant setting. Objective: To determine whether the expression of HIF-1α predicts the relapse of gastric cancer patients who underwent curative surgery followed by adjuvant 5-FU chemotherapy. Materials and Methods: Two HIF-1α knockdown gastric cancer cell lines were established in order to clarify the role of HIF-1α in chemo-resistance against 5-FU. The sensitivity to 5-FU was evaluated by MTT assay, and compared between HIF-1α knockdown cells and control scrambled cells. 5-FU-induced apoptosis was assessed by flow cytometry and the DNA ladder. Furthermore, HIF-1α knockdown xenograft models of nude mice were established to confirm the role of HIF-1α in 5-FU chemo-resistance in vivo. A total of 91 patients with locally advanced gastric cancer who underwent a curative resection were enrolled. Sixty-four of 91 patients received 5-FU based adjuvant chemotherapy after curative surgery. The expression of HIF-1α was immunohistochemically assessed, and correlated with the patient outcome. Results: The inhibition of HIF-1α improved the sensitivity to 5-FU in gastric cancer cell lines, and promoted 5-FU-induced apoptosis in vitro and in vivo. The effect of HIF-1α knockdown on 5-FU-induced apoptosis was more distinctly observed in xenograft tumor than in cell culture. In immunohistochemical analysis, HIF-1α expression was associated with the significantly shorter relapse-free survival and disease-specific survival in all gastric cancer patients (p = 0.045, 0.025), and in the 64 patients in the adjuvant group (p = 0.026, 0.014), but not in the 27 in the surgery group. Multivariate analysis showed that HIF-1α was an independent risk factor for relapse in 64 patients in the adjuvant group (p = 0.029). Conclusions: The current study demonstrated, for the first time, that HIF-1α expression is an independent risk factor for relapse in advanced gastric cancer patients who underwent curative surgery followed by adjuvant 5-FU chemotherapy. A favorable effect of 5-FU might therefore be expected in patients that do not express HIF-1α, whereas, other types of chemotherapy or additional treatments, such as HIF-1α inhibitors, should be considered in patients that do express HIF-1α. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-376.

Abstract LB-86: Analysis of cancer specific gene methylation to diagnose micrometastasis to the lymph nodes and peritoneal cavity in gastric cancer patients

Background and Aims: Aberrant DNA methylation occurs in various cancers and plays important roles in carcinogenesis, tumor progression and chemosensitivity. These epigenetic changes can be utilized to detect micrometastasis in cancer patients. The aim of this study was to confirm that gene methylation is a novel diagnostic marker for micrometastasis to the lymph nodes (LN) and peritoneal fluid (PF) in gastric cancer patients. Methods and Results: 1) An analysis of micrometastasis in lymph nodes: DNA methylation of 7 candidate genes was investigated in 49 primary cancer tissue specimens and corresponding non-cancerous mucosa specimens. Four genes, CHFR, RUNX3, hMLH1, and MGMT, were revealed to express cancer specific methylation and were analyzed in further studies. An analysis of 29 LN that were histologically confirmed as cancer cell positive revealed that 86% of the samples were positive for methylation of at least one of the 4 genes. An analysis of 25 LN that were histologically confirmed to be cancer negative showed 30% of the samples were positive for methylation. In addition, 15% of LN exhibited positive for both methylation and mRNA of CEA, CK19 or CK20. Cancer specific methylation could be detected in the metastatic LN with 86% sensitivity. Micrometastasis was suspected in 30% of LN without histological metastasis. 2) An analysis of micrometastasis in peritoneal fluid: Peritoneal fluid was collected from 80 gastric cancer patients during surgery. DNA methylation of 7 candidate genes was analyzed using the 80 PF samples. The patients were divided into 3 groups; Group A (n = 35): Depth of cancer invasion was less than the muscularis propria (MP), Group B (n = 31): Cancer invasion was beyond the MP. Neither group A nor B showed cancer cells in the peritoneal cytology and histology. Group C (n = 14): Disseminated cancer cells were histocytologically confirmed in the PF. The methylation rate in 3 genes, CHFR, E-cadherin and BNIP3, were significantly different among the groups and increased in order of group A, B and C. Analysis using the multigene methylation in Group C revealed cancer cells in PF with 57% sensitivity and 91% specificity. On the other hand, micrometastasis in PF was suspected in 9% and 19% in Group A and B, respectively. In fact, 2 of the 5 patients with multigene methylation in the group B experienced peritoneal recurrence after the operation, while those without or with a single gene methylation did not experience recurrence. Conclusions: The present study suggests that a methylation analysis in the LN and PF could potentially detect occult neoplastic cells. However, it is important to improve the diagnostic sensitivity by increasing the number of genes that are specifically methylated in cancer cells. Further methylation analyses, by adding several genes, will be discussed at this meeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-86.